2-5A dependent RNase (RNase L, RNase F) is the terminal factor in an interferon-regulated, RNA degradation pathway which has the potential to inhibit both viral and cellular growth. We have cloned and expressed a cDNA which contains the coding sequence of 2-5A dependent RNase. The goals of this project are to perform incisive molecular studies on 2-5A dependent RNase and its gene and to determine the biological significance of the 2-5A system. To obtain basic information about the structure and function of 2-5A dependent RNase, molecular analysis will be performed on the complete protein purified from recombinant cells. To identify the 2-5A binding site this domain will be tagged with a photoaffinity probe, isolated and sequenced. Synthetic peptides and mutant versions of 2-5A dependent RNase will permit identification of which amino acid residues are involved in 2-5A binding and in the inhibition of 2-5A binding after phosphorylation by protein kinase C. To study events controlling 2-5A dependent RNase levels we will monitor transcription during interferon-treatment, growth arrest, and cell differentiation. In addition, the promoter of the 2-5A dependent RNase gene will be isolated, sequenced, and analyzed both for binding to specific nuclear factors and for regulated expression after fusion to a reporter gene. To study the role of the 2-5A system in interferon action we will express sense and antisense RNA for both 2-5A dependent RNase and 2-5A synthetase in mammalian cells. Effects of enhancing or diminishing levels of these enzymes on virus and cell growth will be determined. To downregulate 2-5A dependent RNase levels more efficiently, ribozymes which will specifically cleave mRNA for 2-5A dependent RNase will be expressed in cells allowing effects on interferon action, cell growth, and differentiation to be determined. To establish if mRNA species containing an AU-rich sequence are preferentially cleaved by 2-5A dependent RNase, we will measure breakdown of specific mRNA species in recombinant cells containing enhanced or diminished levels of 2-5A dependent RNase. Because the 2-5A system has the ability to inhibit both virus replication and neoplastic cell growth this project directly impacts on the control of virus infection and cancer in man.